Pre-mRNA cleavage/polyadenylation (C/P) defines the 3' end of a mature transcript. Over half of the human genes have multiple C/P sites (pAs), resulting in mRNA isoforms with different coding sequences (CDS) and/or 3' untranslated regions (3'UTRs). Alternative pA selection (APA) is rapidly recognized as an important layer of gene regulation, affecting protein diversity and mRNA metabolism. The APA pattern of genes varies across cell/tissue types and is dynamically regulated under different conditions, such as development and differentiation. The mechanisms of APA, however, are poorly understood. Our long-term goal is to understand how APA is regulated across cell/tissue types and in development and differentiation. In this grant, we have four Specific Aims (SAs): SA-1, to analyze APA regulation across cell types and in differentiation using newly transcribed RNA; SA-2, to examine how RNA-binding proteins in the C/P complex are involved in APA; SA-3, to examine how recruitment of C/P factors at the promoter affects APA; SA-4, to examine how intronic pAs are regulated. This proposal aims to establish rules of APA regulation at different levels. We expect the results of this grant will provide systematic views on the mechanisms of APA and elucidate its significance in cell functions and in differentiation.